RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Protocol tips
- For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

To detect the expression level of laminin α3, β3, and γ2 chains in A431 cells, total RNA was isolated from cells treated with PBS or 1-2 μM (1 μM for microarray analysis and 2 μM for real-time PCR) LPA using an RNAeasy kit (Qiagen, Germantown, MD). Total RNA digested with RQ RNase-free DNase I (Promega, Madison, WI) and OligoT primer (Roche, Madison, WI) was incubated for 10 min at 70°C, chilled on ice, and added to a 20 ml aliquot of a reaction mixture containing 4 μl of Transcriptor RT reaction buffer (Roche), 1 μl of 10 mM mixture of four dNTPs, and 1 μl of RNasin (Promega). In addition, 0.5 μl of Transcriptor Reverse Transcriptase (Roche, Madison, WI) was added and incubation continued for 1 h at 55°C. To stop the reaction, the reaction mixture was heated for 5 min at 85°C and then chilled on ice. To remove RNA contamination in the reaction mixture, RNase H (Promega) was added and incubated for 30 min at 37°C. RNA integrity was determined by electrophoresis on a 1% agarose gel followed by visualization of intact 18S and 28S ribosomal RNA bands. RNA purity was measured using the A 260/A 280 ratio. To ensure that the optical density at A 260 was in the linear range, various concentrations of RNA were plotted against absorbance.

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Manufacturer protocol

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